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ABSTRACT: Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. PCC 7120, in vivo and in vitro using natural and non-natural carotenoid structures. NSC3 cleaved β-apo-8' -carotenal at 3 positions, C13-C14, C15-C15' , and C13' -C14' , revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4' -diaponeurosporene, 4,4' -diaponeurosporen-4' -al, 4,4' -diaponeurosporen-4' -oic acid, 4,4' -diapotorulene, and 4,4' -diapotorulen-4' -al to generate novel cleavage products (apo-14' -diaponeurosporenal, apo-13' -diaponeurosporenal, apo-10' -diaponeurosporenal, apo-14' -diapotorulenal, and apo-10' -diapotorulenal, respectively). Studying carotenoids with natural or non-natural structures produced using synthetic modules could provide valuable information for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro.
Applied and environmental microbiology 03/2013; · 3.69 Impact Factor
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ABSTRACT: Determination of the complete nucleotide sequence of a cryptic plasmid pMBLT00 from Leuconostoc mesenteriodes subsp. mesenteroides KCTC13302 revealed that it contains 20,721 base pairs (bp), a G + C content of 38.7%, and 18 open reading frames. Comparative sequence and mung been nuclease analyses of pMBLT00 showed that pMBLT00 replicates via the theta replication mechanism. A new, stable E. coli-Leuconostoc shuttle vector pMBLT02, which was constructed from a theta-replicating pMBLT00 replicon and an erythromycin resistance gene of pE194, was successfully introduced into Leuconostoc, Lactococcus lactis, and Pediococcus. This shuttle vector was used to engineer L. citreum 95 to overproduce d-lactate. The L. citreum 95 strain engineered using the plasmid pMBLT02 that overexpresses d-LDH exhibited enhanced production of optically pure d-lactate (61 g/L), 6-times more than that produced by the control strain, when cultured in a reactor supplemented with 140 g/L glucose. Therefore, the shuttle vector pMBLT02 can serve as a useful and stable plasmid vector for further development of a d-lactate overproduction system in other Leuconostoc strains and Lactococcus lactis.
Applied and environmental microbiology 12/2012; · 3.69 Impact Factor
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ABSTRACT: Sucrose is one of the most promising carbon sources for industrial fermentation. We expressed synthetic modules expressing genes of the PEP-PTS and non-PTS pathways in Escherichia coli K12 for comparison. We selected PEP-PTS pathway genes of Lactobacillus plantarum and Staphylococcus xylosus and non-PTS pathway genes of sucrose-utilizing (Scr(+)) E. coli EC3132. Switchable Scr(+) modules expressing E. coli EC3132 non-PTS genes conferred better sucrose-utilizing ability on Scr(-)E. coli K12 than E. coli EC3132. Scr(+) modules expressing S. xylosus PEP-PTS genes conferred a sucrose-utilizing ability on E. coli K12. Among L. plantarum PEP-PTS genes, SacA(LP) and SacK(LP) were functional in E. coli K12. CscA(EC)-CscB(EC)-CscK(EC) (non-PEP-PTS module) or ScrA(SX)-SacA(LP)-SacK(LP) (PEP-PTS module) was introduced to a diapolycopene-producing E. coli strain. In both Scr(+)E. coli K12, the sucrose-utilizing ability of the modules was not affected by diapolycopene formation, indicating that the modular Scr(+) systems could be employed for developing sustainable bioprocesses using sucrose.
Bioresource technology 12/2012; 130C:288-295. · 4.25 Impact Factor
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ABSTRACT: A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules, and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product-3,4,3' ,4' -tetradehydrolycopene-along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.
Applied and environmental microbiology 11/2012; · 3.69 Impact Factor
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ABSTRACT: Osteoarthritis (OA) is the most common rheumatic pathology. One of the major objectives of OA research is the development of early diagnostic strategies such as those using proteomic technology. Synovial fluid (SF) in OA patients is a potential source of biomarkers for OA. The efficient and reliable preparation of SF proteomes is a critical step towards biomarker discovery. In this study, we have optimized a pretreatment method for two-dimensional gel electrophoresis (2DE) separation of the SF proteome, by enriching low-abundance proteins and simultaneously removing hyaluronic acid, albumin, and IgG. SF samples pretreated using this optimized method were then evaluated by 1DE and 2DE separation followed by immunodetection of cartilage oligomeric matrix protein (COMP), a known OA biomarker, and by the identification of 3 proteins (apolipoprotein, haptoglobin precursor, and fibrinogen D fragment) that are related to joint diseases.
Journal of Microbiology and Biotechnology 05/2012; 22(5):654-8. · 1.38 Impact Factor
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ABSTRACT: The biosynthetic pathway for staphyloxanthin, a C(30) carotenoid biosynthesized by Staphylococcus aureus, has previously been proposed to consist of five enzymes (CrtO, CrtP, CrtQ, CrtM, and CrtN). Here, we report a missing sixth enzyme, 4,4'-diaponeurosporen-aldehyde dehydrogenase (AldH), in the staphyloxanthin biosynthetic pathway and describe the functional expression of the complete staphyloxanthin biosynthetic pathway in Escherichia coli. When we expressed the five known pathway enzymes through artificial synthetic operons and the wild-type operon (crtOPQMN) in E. coli, carotenoid aldehyde intermediates such as 4,4'-diaponeurosporen-4-al accumulated without being converted into staphyloxanthin or other intermediates. We identified an aldH gene located 670 kilobase pairs from the known staphyloxanthin gene cluster in the S. aureus genome and an aldH gene in the non-staphyloxanthin-producing Staphylococcus carnosus genome. These two putative enzymes catalyzed the missing oxidation reaction to convert 4,4'-diaponeurosporen-4-al into 4,4'-diaponeurosporenoic acid in E. coli. Deletion of the aldH gene in S. aureus abolished staphyloxanthin biosynthesis and caused accumulation of 4,4'-diaponeurosporen-4-al, confirming the role of AldH in staphyloxanthin biosynthesis. When the complete staphyloxanthin biosynthetic pathway was expressed using an artificial synthetic operon in E. coli, staphyloxanthin-like compounds, which contained altered fatty acid acyl chains, and novel carotenoid compounds were produced, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes.
Journal of Biological Chemistry 04/2012; 287(26):21575-83. · 4.77 Impact Factor
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ABSTRACT: Anaerobiospirillum succiniciproducens requires expensive complex nitrogen sources such as yeast extract and polypeptone for its growth and succinic acid production.
It was found thatA. succiniciproducens was able to grow in a minimal medium containing glucose when supplemented with corn steep liquor (CSL) as the sole complex
nitrogen source. The concentration of CSL had a significant effect on the glucose consumption byA. succiniciproducens. When 10–15 g/L of CSL was supplemented, cells were grown to an OD660 of 3.5 and produced 17.8 g/L succinic acid with 20 g/L glucose. These results are similar to those obtained by supplementing
yeast extract and polypeptone, thereby suggesting that succinic acid can be produced more economically using glucose and CSL.
Biotechnology and Bioprocess Engineering 04/2012; 5(5):379-381. · 1.28 Impact Factor
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ABSTRACT: The effects of three phosphoenolpyruvate (PEP)-dependent PTS carbon sources (glucose, mannose and maltose) and three non-PTS
carbon sources (glycerol, galactose, and lactose) on the formation of four carotenoids with diverse structures and on the
cell growth of the recombinant Escherichia coli were investigated. The biosynthetic pathways of four carotenoids, C30 diapolycopene, C30 diapotorulene, C40 lycopene, and C40 beta-carotene, were engineered in E. coli. The resulting E. coli cells were grown in a mineral medium supplemented with each of the six carbon sources. Among the six carbon sources, non-PTS
glycerol showed the highest performance in production of all four carotenoid structures, whereas PTS glucose showed the lowest
performance. Based on the conversion yield, carotenoid-producing capability, and the cell density, we found that there was
no close correlation between PTS and non-PTS transport mechanism and carotenoid formations in E. coli.
KeywordsCarotenoids–Carbon source–
E. coli
–Metabolic engineering
World Journal of Microbiology and Biotechnology 04/2012; 26(12):2231-2239. · 1.53 Impact Factor
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ABSTRACT: ApckA gene encoding phosphoenolpyruvate carboxykinase (PEPCK) was cloned and sequenced from the succinic acid producing bacteriumMannheimia succiniciproducens MBEL55E. The gene encoded a 538 residue polypeptide with a calculated molecular mass of 58.8 kDa and a calculated pI of 5.03.
The deduced amino acid sequence of theM. succiniciproducens MBEL55E PEPCK was similar to those of all known ATP-dependent PEPCKs.
Biotechnology and Bioprocess Engineering 04/2012; 7(2):95-99. · 1.28 Impact Factor
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Pyung Cheon Lee
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ABSTRACT: Cellular targeting of biosynthetic pathway enzymes is an invaluable technique in metabolic engineering to modify metabolic fluxes towards metabolite of interest. Especially, recombinant carotenoid biosynthesis in yeasts should be balanced with a precursor pathway present in a specific cellular location because yeasts, being eukaryotes, have more defined intracellular location. Here, peroxisomal targeting of lycopene pathway enzymes, CrtE, CrtB, and CrtI, by fusing to peroxisomal targeting sequence 1 (PTS1) in Pichia pastoris X-33 is described.
Methods in molecular biology (Clifton, N.J.) 01/2012; 898:161-9.
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ABSTRACT: Algae biomass is a potential raw material for the production of biofuels and other chemicals. In this study, biomass of the marine algae, Ulva lactuca, Gelidium amansii,Laminaria japonica, and Sargassum fulvellum, was treated with acid and commercially available hydrolytic enzymes. The hydrolysates contained glucose, mannose, galactose, and mannitol, among other sugars, at different ratios. The Laminaria japonica hydrolysate contained up to 30.5% mannitol and 6.98% glucose in the hydrolysate solids. Ethanogenic recombinant Escherichia coli KO11 was able to utilize both mannitol and glucose and produced 0.4g ethanol per g of carbohydrate when cultured in L. japonica hydrolysate supplemented with Luria-Bertani medium and hydrolytic enzymes. The strategy of acid hydrolysis followed by simultaneous enzyme treatment and inoculation with E. coli KO11 could be a viable strategy to produce ethanol from marine alga biomass.
Bioresource technology 08/2011; 102(16):7466-9. · 4.25 Impact Factor
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ABSTRACT: Menaquinone-8 (MK-8, vitamin K) is composed of a non-polar side chain and a polar head group. Escherichia coli was chosen and metabolically engineered as a microbial platform for production of MK-8. MK-8 content in E. coli was significantly enhanced by modulating two precursor pools, which supply a non-polar side chain and a polar head group, and further increased by blocking formation of the competitor ubiquinone-8 (Q-8). Overexpression of E. coli IspA, DXR, or IDI increased MK-8 content up to twofold. A similar positive effect was also observed when E. coli MenA, MenB, MenC, MenD, MenE, MenF, or UbiE was overexpressed. The Q-8-deficient ubiCA mutant enhanced MK-8 content by 30% compared to wild-type E. coli. When MenA or MenD was overexpressed, MK-8 content was enhanced fivefold compared with wild-type E. coli.
Biotechnology and Bioengineering 03/2011; 108(8):1997-2002. · 3.95 Impact Factor
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ABSTRACT: Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and β-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5'-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL⁻. The expression level of hGH was further enhanced, up to ~42% of the TCP, by adding the N-terminal peptide tag of β-galactosidase to hGH, which was comparable to the expression of ~43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant β-tyrosinase was successfully expressed at a rate of up to ~45% of the TCP in pRBS(fnr) in W3110narL⁻. From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.
Journal of biotechnology 01/2011; 151(1):102-7. · 2.88 Impact Factor
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ABSTRACT: Seven Escherichia coli strains, which were metabolically engineered with carotenoid biosynthetic pathways, were systematically compared in order to investigate the strain-specific formation of carotenoids of structural diversity. C30 acyclic carotenoids, diaponeurosporene and diapolycopene were well produced in all E. coli strains tested. However, the C30 monocyclic diapotorulene formation was strongly strain dependent. Reduced diapotorulene formation was observed in the E. coli strain Top10, MG1655, and MDS42 while better formation was observed in the E. coli strain JM109, SURE, DH5a, and XL1-Blue. Interestingly, C40 carotenoids, which have longer backbones than C30 carotenoids, also showed strain dependency as C30 diapotorulene did. Quantitative analysis showed that the SURE strain was the best producer for C40 acyclic lycopene, C40 dicyclic β-carotene, and C30 monocyclic diapotorulene. Of the seven strains examined, the highest volumetric productivity for most of the carotenoids structures was observed in the recombinant SURE strain. In conclusion, we showed that recombinant hosts and carotenoid structures influenced carotenoid productions significantly, and this information can serve as the basis for the subsequent development of microorganisms for carotenoids of interest.
Applied biochemistry and biotechnology 12/2010; 162(8):2333-44. · 1.94 Impact Factor
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ABSTRACT: The spheroidene monooxygenase CrtA of Rhodobacter sphaeroides introduces a keto group and/or hydroxy group at the ends of nonnative substrates in Escherichia coli, resulting in the production of novel oxocarotenoids. The heme-containing CrtA is not a P450 enzyme but a new type of oxygenase.
Applied and environmental microbiology 11/2010; 76(21):7328-31. · 3.69 Impact Factor
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ABSTRACT: In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY(2) and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed beta-carotene pathway synthesized an unexpected 7,8-dihydro-beta-carotene in addition to beta-carotene. Extension of the beta-carotene pathway with the B. linens beta-ring desaturase CrtU and Pantoea beta-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.
Applied and environmental microbiology 08/2010; 76(15):5199-206. · 3.69 Impact Factor
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ABSTRACT: Succinic acid-producing Anaerobiospirillum succiniciproducens was anaerobically grown in glucose-fed continuous cultures using glucose as a carbon source, and the metabolic flexibility of A. succiniciproducens in response to varying glucose concentrations and dilution rates was examined. Both succinic acid (SA) and acetic acid (AA) formation was growth-associated, and their growth-rate-related coefficients (KSA/X, KAA/X) and nongrowth-rate-related coefficients (K'SA/X, K'AA/X) were slightly influenced by glucose concentrations. A high glucose concentration (38 g/l) and high growth rate (0.63 h-1) did not induce by-product formation.
Journal of Microbiology and Biotechnology 11/2009; 19(11):1379-84. · 1.38 Impact Factor
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ABSTRACT: Cellular targeting of lycopene biosynthetic enzymes was investigated in Pichia pastoris X-33. Three lycopene pathway enzymes, CrtE, CrtB, and CrtI, were fused to fluorescent EGFPs with or without a peroxisomal targeting sequence (PTS1) and then expressed in P. pastoris. When P. pastoris was grown in YPD, the PTS1 fusion enzymes were found to be localized in peroxisomes, whereas the enzymes not fused with PTS1 were equally distributed throughout the entire cell. A similar targeting pattern was also observed in P. pastoris strains that were grown in peroxisome-proliferating medium, YPOT. Analysis of the fluorescent images of isolated peroxisomes showed that the PTS1 fused enzymes were dominantly present in peroxisomes whereas small amount of the enzymes not fused with PTS1 were non-specifically sent to peroxisomes. These results indicate that PTS1 specifically target lycopene pathway enzymes into peroxisomes and this targeting pathway was strong enough to overcome their inherent targeting program. In conclusion, we first showed that carotenogenic enzymes can be targeted into the specific cellular location of recombinant hosts and this targeting strategy can serve as the basis for the subsequent development of sophisticated pathway engineering in microorganisms.
Journal of biotechnology 04/2009; 140(3-4):227-33. · 2.88 Impact Factor
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ABSTRACT: Succinic acid-producing Anaerobiospirillum succiniciproducens was anaerobically grown on galactose, galactose/glucose, or galactose/lactose in order to study its galactose fermentation. Unlike a previous report, A. succiniciproducens was found to efficiently metabolize galactose as the sole carbon source at a rate of 2.4 g/g-DCW/h and produced succinic acid with as high a yield of 87% as with using glucose. When glucose and galactose were present, A. succiniciproducens metabolized both sugars simultaneously. Furthermore, when lactose and galactose coexisted, lactose did not inhibit the galactose fermentation of A. succiniciproducens. Therefore, co-utilization of galactose and other sugars can improve the productivity and economy of bio-based succinic acid processes.
Journal of Microbiology and Biotechnology 12/2008; 18(11):1792-6. · 1.38 Impact Factor
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ABSTRACT: Enzymatic steps from two different biosynthetic pathways were combined in Escherichia coli, directing the synthesis of a new class of biomolecules--ubiquinones with prenyl side chains containing conjugated double bonds. This was achieved by the activity of a C(30) carotenoid desaturase, CrtN, from Staphylococcus aureus, which exhibited an inherent flexibility in substrate recognition compared to other carotenoid desaturases. By utilizing the known plasticity of E. coli's native ubiquinone biosynthesis pathway and the unusual activity of CrtN, modified ubiquinone structures with prenyl side chains containing conjugated double bonds were generated. The side chains of the new structures were confirmed to have different degrees of desaturation by mass spectrometry and nuclear magnetic resonance analysis. In vivo (14)C labeling and in vitro activity studies showed that CrtN desaturates octaprenyl diphosphates but not the ubiquinone compounds directly. Antioxidant properties of conjugated side chain ubiquinones were analyzed in an in vitro beta-carotene-linoleate model system and were found to be higher than the corresponding unmodified ubiquinones. These results demonstrate that by combining pathway steps from different branches of biosynthetic networks, classes of compounds not observed in nature can be synthesized and structural motifs that are functionally important can be combined or enhanced.
Applied and environmental microbiology 10/2008; 74(22):6908-17. · 3.69 Impact Factor
